Stabilization of plasma with invert sugar



Unite This invention relates to the processing of proteinaceous materials and has particular reference to a process for the stabilization of proteins to heat.

It is desirable to be able to heat proteinaceous materials such as human blood plasma in order to inactivate the virus of homologous serum hepatitis. The best evidence to date, based upon experience with albumin and the addition of virus-infected plasma to albumin, is that heating of the plasma for hours at 60 C. does inactivate the virus. Unfortunately, plasma contains certain proteins which are readily denatured by heat and which tend either to precipitate or to form solutions of increased viscosity and turbidity. In the process of heating, the denaturing proteins may form complexes of unknown nature with some of the other plasma proteins which are not intrinsically denatured under such conditions of heating.

Accordingly, the primary object of this invention is to provide a process for the production of heat-stable human blood plasma and other proteins, and products resulting from such a process.

The protein fractions of plasma that are the most sensitive to heat are the lipoproteins and fibrinogen. The former are readily removed by conventional techniques; it is the latter which provides some considerable difficulty. Another object of this invention is, therefore, to provide a process for the production of heat-stable human blood plasma wherein the more heat-sensitive proteins are removed and wherein those remaining are rendered heatstable.

When blood is drawn for plasma in sodium citrate solution, a reaction occurs between the calcium of the blood and the citrate which effectively removes the calcium. Calcium is necessary for the conversion of prothrombin to thrombin, and the latter is necessary for the conversion of fibrinogen to fibrin (which is responsible for the clotting of blood). Thus, as the blood is drawn and mixed with citrate, the calcium is removed and clot formation is prevented. However, there appear to be some changes occurring in some of the fibrinogen molecules that might be viewed as intermediate stages in the transformation to fibrin. These intermediate, half-way clotted fibrinogen molecules could be reasonably viewed as those most readily subject to precipitation by heat or other conditions, such as standing, agitation, freezing, etc.

Partial to nearly complete defibrination and complete delipoproteinization can be achieved in various Ways, such as by the freezing and thawing of plasma and filtration. In the process of stabilization of the remaining proteins, described hereafter, it is important that the preliminary treatment of this kind be sufiicient to remove the labile fibrinogen although it is not essential or even desirable that the plasma be completely defibrinated. If, in addition, the plasma is subjected to the exposure treatment described in the copending application of Calver O. Mace, Serial Number 532, 769, filed September 6, 1955, now abandoned, on Process for the Treatment of Human Blood Plasma, then the stability of all components to heat is further increased.

Removal of labile fibrinogen and lipoproteins is desirable for added reasons over and above heat stability. These labile substances tend to precipitate during storage or shipment, so that in the ordinary course of events, the

States Patent "ice plasma actually injected into a patient does not contain these substances (the precipitates are removed by a filter in the administration set). Finally, plasma that is opalescent to turbid because of aggregation of these substances bears a striking resemblance to contaminated plasma. Thus, usable plasma might be discarded where wrongly thought to be contaminated or, worse, contaminated plasma might be used inadvertently thinking it merely con tained fibrin precipitate.

Therefore, it is another object of this invention to provide a human blood plasma for which the lipoproteins and the least necessary amount of fibrinogen have been removed, which is stable to heat and agitation in the fluid state, which has minimal alterations of viscosity and opalescence, and which contains concentrations of the important electrolytes most closely approaching the physiological range; and to provide a process for producing such a plasma.

Another object of this invention is to' provide a process for the preparation of a stabilizer for human blood plasma.

Another object of this invention is to provide a process of the type described generally above, which is adapted to be carried out inexpensively, utilizing conventional equipment.

Other objects and advantages of this invention it is believed will be readily apparent from the following detailed description of preferred embodiments thereof.

Briefly, this invention includes the discovery that proteins such as human blood plasma are rendered heat and agitation stable by the addition thereto of heated invert sugar containing minor proportions (from about 0.01% to about 2.0% from a practicable, commercial standpoint) of levulinic acid or a physiologically acceptable salt thereof. Preferably, the plasma is first treated to remove the more labile fibrinogen components and the lipoproteins by conventional methods such as freezing, thawing and filtration, leaving in solution some of the more stable fibrinogen. It has been found that the heat stability of plasma is significantly enhanced by the addition thereto of levulinic acid or its salts alone in amounts from about 1% to about 4% (wgt./vol., liquid plasma); by theaddition of mixtures of unheated invert sugar containing from about 0.1% to about 5% of levulinic acid or its salts either naturally present or added as such; and by the addition of heat-sterilized invert sugar alone, since one of the products formed by heating of invert sugar solutions is levulinic acid.

However, optimum results are obtained by adding to liquid plasma a stabilizing agent prepared by heat sterilizing (i.e., autoclaving at pressures from 5 to pounds per square inch for time periods from about 3 hours to about 15 minutes) a concentrated aqueous solution of invert sugar to which has been added a trace amount of levulinic acid or a salt (such as sodium, potassium, calcium, ammonium, etc.) thereof. The resulting stabilized plasma may then be heat-treated by the 10-hour, C. method to render it hepatitis-free, and this heated plasma may be used as such or dried for later reconstitution and use. Best results from the standpoint of drying and reconstitution are obtained if the plasma is treated in accordance with the method of said copending application Serial No. 532,769, prior to addition of the stabilizing agent. The proportion of stabilizing agent to liquid plasma falls within the range of 1-20% (wgt./vol.), based upon the dry weight of the invert sugar prior to heating. Although somewhat less stabilizer may be used if the plasma is first treated with the method of said copending application Serial No. 532,769, the preferred amount of added invert sugar-levulinic acid stabilizer is from about 2.5 to about 5 percent on a dry, unheated sugar basis.

The following specific examples are illustrative of the process of this invention, but it is not intended to limit the invention thereto:

Example 1 The stabilizing agent was prepared by making up a 50% solution of invert sugar in distilled water. To this solution, which had a pH of about 3.0, was added 0.02% levulinic acid, and the mixture was autoclaved at a pressure of 25 pounds per square inch for about 1 hour. On cooling to room temperature, the heated solution was adjusted to a pH of 11.0 with a saturated solution of sodium hydroxide and the pH then brought back to 8.0 with concentrated (36%) hydrochloric acid.

25 liters of normal human blood plasma, collected in accordance with NIH requirements, in 5% sodium citrate solution was pooled and frozen. 2.5 liters of this frozen plasma was thawed at 27 C., re-frozen and then again thawed at 27 C. The thawed plasma was drawn off aseptically into a conventional 12 liter jug containing an aspirator tube, and was filtered through a #2 asbestos filtration pad under positive nitrogen pressure. The filtrate was run through a #6 filter pad again under positive nitrogen pressure, and the filtrate held for 5 days at a temperature of 27 C.

The stored filtrate was then gently agitated to aggregate precipitated fibrin on the outer surface of the aspirator tube, and then run through a #8 filter pad under positive nitrogen pressure. The filtrate was double irradiated by passing it twice in a thin film at the rate of 250 cc./min. through a Michael-Reese Ultraviolet Centrifugal Filmer (US. Patent No. 2,452,201) utilizing ultraviolet light of 2537 Angstroms.

One part of the heated invert sugar-levulinic acid stabilizer solution was then added to nine parts of the double-irradiated plasma (222.2 cc. of stabilizer to 2 liters of plasma) to provide 5% of the sugar solution, on a dry sugar basis. The mixture was filtered through an E0 sterilizing grade asbestos filter pad into evacuated sterile containers to produce the final filtrate which is ready for the hepatitis virus-destroying heat treatment. It should be noted that all filtration and other steps were carried out in the cold (2-7 C). The filter pads were manufactured by the Eitel Engineering Corp.

In order to test the stabilized plasma, samples were placed in 30 cc. containers and these were heated in a water bath for 10 hours at 60 C. There was only a trivial increase, after heating, in viscosity and in opalescence as determined by the Coleman nephelocolorimeter. Electrophoretic studies (both Tiselius and paper) showed no major alterations of the plasma proteins caused by the heating. Electrolyte studies showed essentially normal electrolytes except for the higher-than-normal sodium which was due to the sodium citrate originally added to the blood. Samples of the heated plasma were injected intraperitoneally into mice (Olitsky strain) in amounts equalling approximately 8% of body weight without apparent injury. Other samples were injected into 6 humans intraperitoneally in amounts of ml. without subjective or objective reactions.

The liquid plasma stabilized in accordance with the present invention may be dried by conventional lyophilization procedures after heat treatment, for later reconstitution and use.

The following example illustrates the use of the process in combination with the process of said copending application Serial No. 532, 769.

Example 2 liters of normal human blood plasma was collected, frozen, thawed, filtered and double-irradiated, all in accordance with Example 1. The irradiated plasma was filtered through an E0 sterilizing grade asbestos filter pad into 600 cc. units in evacuated sterile containers. The units were shell-frozen and lyophilized (freeze dried) over a period of about 42 hours. The containers were provided with sterile cotton plugs and exposed by placing them in a water bath at 45 C. for 2 days under a relative humidity of 55%.

Each of the dried and exposed plasma units was then reconstituted to 600 cc., cooled to 2-7" C. and filtered through a #9 pad. 51 cc. of stabilizer solution prepared in accordance with Example 1 was added to each 600 cc. unit, to provid 4% of the stabilizer solution, on a dry sugar basis. The mixtures were then filtered through an E0 pad and filled into final containers. These were placed in a water bath for 10 hours at 60 C. to undergo the hepatitis virus-destroying heat treatment. The treated material was suitable for use at this point. However, for convenienc it was then shell-frozen and lyophilized to produce a dried plasma which was readily reconstitutable for use as desired.

In carrying out the process of this invention it should be noted that the addition of the stabilizer should be done in neutral or alkaline solution. The sugar-levulinic acid solution may be heated in acid solution as well as in alkaline, but an acid solution is preferred because there is a lesser formation of non-contributory colored components in acid. In addition to conferring increased stability to heat, the process of thi invention imparts increased agitation stability to the plasma.

The use of invert sugar is desirable in addition to its stabilization effect, since it is more readily used for energy than is glucose and has a greater nitrogen sparing action. In any event, both glucose and fructose are desired in most cases where plasma is clinically indicated. In addition, there is evidence that fructose is superior to glucose in certain toxic states and in certain metabolic disorders.

In addition to its use in the treatment of human blood plasma, the process of thi invention is applicable to the stabilization of other proteins, such as, for example, bovine albumin, gamma globulin, and the like.

Having fully described our invention, it is to be understood that we do not wish to be limited to the details set forth, but our invention is of the full scope of the appended claims.

We claim:

1. In a process for the production of heat and agitation stable human blood plasma, the steps comprising physically removing lipoproteins and labile fibrinogens from liquid plasma, and adding to said plasma a stabilizing agent comprising a heat sterilized aqueous solution of invert sugar, said solution comprising between about 1 and about 20% of the plasma-stabilizing agent admixture, based upon the dry weight of the invert sugar prior to heat sterilization thereof.

2. In a process for the production of heat and agitation stabl human blood plasma, the steps comprising physically removing by freezing, thawing and filtration techniques lipoproteins and labile fibrinogens from liquid plasma, and adding to said plasma a stabilizing agent comprising a heat sterilized aqueous solution of invert sugar, said solution comprising between about 1 and about 20% of the plasma-stabilizing agent admixture, based upon the dry weight of the invert sugar prior to sterilization thereof.

3. In a process for the treatment of dried human blood plasma, th steps comprising subjecting the dried plasma to the action of air at a temperature and for a length of time sufficient to insolubilize labile protein fractions contained in said plasma, reconstituting said plasma with distilled water, separating the insolubilized fractions from the solution, and adding to the solution a stabilizing agent comprising a heat sterilized aqueous solution of invert sugar, said solution comprising between about 1 and about 20% of the plasma-stabilizing agent admixture, based upon the dry weight of the invert sugar prior to heat sterilization thereof.

4. In a process for the production of heat and agitation stable human blood plasma, the steps comprising physically removing lipoproteins and labile fibrinogens from liquid plasma, drying said plasma, subjecting the dried plasma to the action of air at a temperature and for a length of time suflicient to insolubilize labile protein fractions contained in said plasma, reconstituting said plasma with distilled water, separating the insolubilized fractions from the solution, and adding to the solution a stabilizing agent comprising a heat sterilized aqueous solution cf invert sugar, said solution comprising between about 1 and about 20% of the plasma-stabilizing agent admixture, based upon the dry Weight of the invert sugar prior to heat sterilization thereof.

5. In a process for the treatment of dried human blood plasma, the steps comprising reconstituting said plasma with distilled water, separating the insolubilized fractions from the solution, and adding to the solution a stabilizing agent comprising a heat sterilized aqueous solution of invert sugar, said solution comprising between about 1 and about 20% of the plasma-stabilizing agent admixture, based upon the dry Weight of the invert sugar prior to heat sterilization thereof.

6. In a process for the treatment of dried human blood plasma, the steps comprising reconstituting said plasma with distilled Water, separating the insolubilized fractions from the solution, and adding to the solution a minor effective amount of a stabilizing agent comprising a heat sterilized aqueous solution of invert sugar.

7. In a process for the treatment of dried human blood plasma, the steps comprising subjecting the dried plasma to the action of air at a temperature and for a length of time sufiicient to insolubilize labile protein fractions contained in said plasma, reconstituting said plasma With distilled water, separating the insolubilized fractions from the solution, and adding to the solution a minor effective amount of a stabilizing agent comprising a heat sterilized aqueous solution of invert sugar.

References Cited in the file of this patent FOREIGN PATENTS Great Britain Feb. 18, 1953 OTHER REFERENCES Chemical Abstracts, vol. 32, 1938, page 6288.

MacKenzie: Sugars and Their Simple Derivatives, page 186, pub. by Gurney & Jackson, London, England, 1913.

Boyer: J. Biol. Chem., 1946, pages 181-198.

Powell et al.: J. Am. Pharm. Assoc, vol. 37, February 1948, pages -70, page 66 is especially pertinent.

Scatchard et al.: J. Clin. Inv., vol. 23, No. 4, July 1944, pages 445-453.

Reid et al.: Am. J. Clin. Path., January 1949, vol. 19, No. 1, pages 10-15.

Pennell: Am. J. Pharm., May 1952, vol. 124, No 5, pages 154-160. 

1. IN A PROCESS FOR THE PRODUCTION OF HEAT AND AGITATION STABLE HUMAN BLOOD PLASMA, THE STEPS COMPRISING PHYSICALLY REMOVING LIPOPROTEINS AND LABILE FIBRINOGENS FROM LIQUID PLASMA, AND ADDING TO SAID PLASMA A STABILIZING AGENT COMPRISING A HEAT STERILIZED AQUEOUS SOLUTION OF INVERT SUGAR, SAID SOLUTION COMPRISING BETWEEN ABOUT 1 AND ABOUT 20% OF THE PLASMA-STABILIZING AGENT ADMIXTURE, BASED UPON THE DRY WEIGHT OF THE INVERT SUGAR PRIOR TO HEAT STERILIZATION THEREOF. 